Effect of eNOS gene G894T mutation on the ability of vascular endothelial cells to produce nitric oxide and its significance
10.3760/cma.j.issn.1008-1372.2019.05.010
- VernacularTitle:eNOS基因G894T变异对血管内皮细胞产生一氧化氮能力的影响及其意义
- Author:
Liwen ZHENG
1
;
Wanchun WANG
;
Xinzhan MAO
;
Jiangdong NI
;
Jianwei WEI
;
Ding CHEN
;
Ding LI
;
Minzhi MAO
Author Information
1. 中南大学湘雅二医院骨科
- Keywords:
Endothelium,vascular;
Nitric oxide synthase;
Polymorphism,single nucleotide;
Femur head necrosis
- From:
Journal of Chinese Physician
2019;21(5):677-681,687
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the relationship between vascular endothelial cell nitric oxide synthase (eNOS) gene polymorphism and the pathogenesis of avascular femoral head necrosis (ANFH).Methods The eNOS full-length CDS fragments,containing 894G or 894T separately,was subcloned into lentiviral expression vector,and then infect the human umbilical vein endothelial cells (HUVEC).The contents of nitric oxide NO and cyclic guanosine monophosphate (cGMP) in cell culture supernatant were detected in a time-dependent manner.The luciferase-labeled reporter plasmid pNF-κB-luc was co-transfected with the reference control plasmid pRL-TK into HUVEC cells infected with LV-eNOS-894G,LV-eNOS-894T,and control lentivirus (LV-NC) for 48 h.The luciferase activity of each group was detected.The expression of nuclear factor (NF)-κB protein and eNOS protein in HUVEC cells were detected by Western blot assay.The HUVEC cells in each group were co-cultured with hFOB1.19 cells,and the concentration of alkaline phosphatase (ALP) or osteocalcin (OCN) in the supernatant were detected at different time points.Results The contents of NO and cGMP in the cell culture supernatant of the full-length lentivirus expressing eNOS gene (containing 894G or 894T) were significantly higher than that in the empty cell group and the empty vector group,and the contents of NO and cGMP in the cell culture supernatant of the 894G group were significantly higher than that of 894T group (P ＜ 0.01).Compared with blank cells,the expression levels of NF-κB/p65 protein and eNOS protein were significantly increased in cells expressing eNOS,and the expression of NF-κB/p65 protein in 894G group was significantly increased than 894T group,but there was no difference in eNOS protein expression between the 894G and 894T groups;each group of HUVEC cells were co-cultured with hFOB1.19 osteoblasts,and at each of the same time points,the concentrations of ALP and OCN in the cell culture supernatant expressing lentivirus were significantly higher than that in the empty cell group and the empty vector group,and the ALP and OCN concentrations in the cell culture supernatant of the 894G group were significantly higher than those in the 894T group.Conclusions The eNOS gene exon G894T mutation reduces the levels of nitric oxide and cGMP produced by vascular endothelial cells through the eNOS-NO pathway,affecting the expression levels of NF-κB/p65 protein and eNOS protein,and reducing osteoblast activity,and blood supply to blood vessels.It may be one of the pathogenic mechanisms leading to femoral head necrosis.
